1. Field of the Invention
The present invention relates, in general, to methods and compositions for controlling insects in monocotyledonous plants (monocots), particularly maize. More precisely, the present invention relates to (1) a method for controlling insects comprising feeding or contacting an insect with an insecticidal amount of transgenic monocotyledonous plant cells comprising recombinant DNA comprising a coding sequence encoding peroxidase and (2) a fertile transgenic monocot plant comprising recombinant DNA comprising a coding sequence encoding peroxidase.
2. Background Information
Insect pests are a major factor in the loss of the world's commercially important agricultural crops. Broad spectrum chemical pesticides have been used extensively to control or eradicate pests of agricultural importance. Although insecticides have been effective in controlling most harmful insects, there are considerable problems associated with the use of these compounds. Insecticides are expensive and costly to apply. Often repeated applications are necessary for effective control. There is also concern that insects have or will become resistant to many of the chemicals used in controlling them. Insecticides often kill beneficial insects which are pollinators or prey on the herbivorous insects. Additionally, there are environmental hazards associated with the long term use of chemical insecticides.
Programs of pest management are being introduced which lower the use of chemical insecticides. These programs include the improvement of crops by selection, the employment of biological control agents and insect predators, and the incorporation of insect resistant genes through breeding programs and genetic engineering. The most widely utilized genes for genetic engineering are the crystal protein genes from Bacillus thuringiensis. See, for example, Rice et al., EP 292,435 (to Ciba-Geigy AG) and Koziel et al., WO 93/07278 (to Ciba-Geigy AG). The majority of the crystal proteins made by Bacillus are toxic to larvae of insects in the orders Lepidoptera, Diptera and Coleoptera. In general, when an insecticidal crystal protein is ingested by a susceptible insect, the crystal is solubilized and acts as a toxic moiety. To avoid the development of insects which are resistant to these toxins, additional toxins are needed which have additive or synergistic affects.
Peroxidases are a subclass of oxido-reductases that use a peroxide such as H.sub.2 O.sub.2 as an oxygen acceptor. Peroxidases are heme-containing monomeric glycoproteins able to bind divalent cations (mainly Ca.sup.2+, but also Mn.sup.2+) (Maranon and Van Huystee, Phytochemistry 37: 1217-1225 (1994)). The prosthetic groups for peroxidase have different roles. While the heme group is involved in catalysis, the divalent cations stabilize the heme moiety, and the glycosyl groups may help to stabilize the peroxidase by decreasing its turnover rate (Maranon and Van Huystee, Phytochemistry 37: 1217-1225 (1994)).
Peroxidases are often grouped into anionic, cationic, and neutral forms according to their migration on isoelectric focusing gels. Although as enzymes they are considered to have wide substrate specificity, they do appear to have some substrate "preferences" for different isoenzymes (Van Huystee, Ann. Rev. Plant Phyisiol., 205-219 (1987)). There are several types of peroxidases and related enzymes including guaiacol peroxidase, NADH peroxidase, cytochrome-C peroxidase, catalase, glutathione peroxidase, L-ascorbate peroxidase, and manganese peroxidase.
In plants, peroxidases are monomeric proteins which are highly complex enzymes whose activities are closely regulated by the plant. Peroxidases are critical in the biosynthesis of plant cell walls. Peroxidases promote the peroxidative polymerization of the monolignols coniferyl, .rho.-coumaryl, and sinapyl alcohol into lignin (Greisbach, In: The Biochemistry of Plants, Ed. Conn, Academic, New York pp. 457-480 (1991)). Different plant species have varying ratios of the monolignol species assembled in a semi-random fashion (Hwang et al., Carbohydrate Polymers 14:77-88 (1991)). Lignification serves to strengthen and reinforce cell walls. The overall result is a toughening of the plant tissue.
A tobacco anionic peroxidase was utilized to transform N. tabacum and N. sylvestris (Lagrimini, Plant Cell 2:7-18 (1990); Lagrimini, Plant Physiology 96:577-583 (1991)). These transgenic plants constitutively overexpressed a tobacco anionic peroxidase from a 35S promoter. The most striking phenotype of peroxidase overexpression was chronic wilting which begins at approximately the time of flowering. In addition, the plants were retarded in growth, had smaller, compacted cells, and brown rapidly in response to wounding.
The same construct was also utilized to transform tomato plants (Lagrimini et al., J Am. Soc. Hort. Sci. 117:1012-1016 (1992); Lagrimini et al., Hortscience 28:218-221(1993)). These plants were also found to wilt severely after flowering, and showed excessive browning and reduced fruit size.
Initial studies have shown that some tissues of transgenic tobacco and tomato plants expressing a tobacco anionic peroxidase gene were resistant to some insects (Dowd et al., presentation at the National Meeting of the Entomological Society of America, Indianapolis, December 1993). Tobacco and tomato are closely related dicots belonging to the same family, the Solanaceae.
In contrast, the transgenic monocots of the present invention have vastly different physiology, biochemistry, anatomy, and metabolism when compared to dicots. For example, monocots have different codon usage, use C4 instead of C3 metabolism, have different fatty acid content, imperfect flowers, and the like. Thus, it was unknown whether substrates would exist in monocots that could be used by peroxidase to control insects.
Further, peroxidases are glycoproteins that must undergo specific post-transcriptional modification and incorporation of heme-containing groups to be stable and enzymatically active. Peroxidases are involved in the synthesis of secondary metabolites and lignins whose nature depends on the substrates available in the specific plant. Therefore, the final products obtained by expressing peroxidases may differ from plant to plant.
Additionally, resistance to corn earworms is negatively correlated to silk browning indicating that an increase in peroxidase would lower resistance (Byrne et al., Environ. Entomol. 18:356-360 (1989)). This teaches away from using peroxidase to control insects in monocots.
Further, altered lignin production in corn (in bm mutants) causes increased susceptibility to insects (Barriere and Argillier, Agronomie 13:865-876 (1993)). Thus, a foreign peroxidase which alters lignification would not be expected to decrease susceptibility to insects. Further, it was unexpected from the teachings of Bergvinson et al., The Canadian Entomologist 127:111-122 (January/February 1995) that insect resistance is imparted to plants by toughening of tissues due to peroxidase activity in the early stages of growth.
Therefore, prior to the present invention, the effect of expressing a recombinant peroxidase in monocots was unpredictable.